Investigating the Efficiency of Photothrombosis-associated Focal Cerebral Ischemia by Monitoring Histopathological Changes and Neurobehavioral Performance in a Model of Mouse

Abstract

Background Photothrombotic (PT) stroke model is a novel method to induce ischemic stroke in the target site using excitation of a photosensitive agent such as Rose Bengal dye after light illumination. Here, we performed a photothrombotic-induced brain ischemic model using green laser and photosensitive agent Rose Bengal (RB) and its efficiency evaluated through cellular, histological, and neurobehavioral approaches.Methods Mice were randomly allocated into RB; Laser irradiation; and RB + Laser irradiation groups. Mice were exposed to green laser at a wavelength of 532 nm and intensity 150 mW in a mouse model after injection of RB. Four circular polarizers were applied to control output beam intensity. The laser was illuminated on the target region under stereotactic surgery. Mice were euthanatized after 28 days. We assessed ischemic stroke outcomes using multiple approaches including macroscopic, microscopic (H&E and Cresyl violet staining). The volume of lesion sites was calculated using unbiased stereological assay and Cavalier’s principle. We performed double immunofluorescence staining for NeuN and BrdU on day 28. To assess the quality of ischemic stroke, neurological behavior, and functional deficits were done on days 1, 7, 14, and 28. Results Laser irradiation plus RB promoted pale ischemic changes after 7 days. Microscopic staining revealed neural tissue degeneration and demarcated necrotic site and neuronal injury plus prominent astrogliosis in the periphery of irradiated sites on day 28. NeuN+/BrdU+ staining showed a significant number of proliferating cells in Laser irradiation plus RB group compared to the group (p<0.05) while the percent of NeuN+ cells reduced. Neurological behavior and functional deficits were detected in mice from Laser irradiation plus the RB group. No histological or functional deficits were detected in RB and Laser irradiation groups. Conclusion Our study revealed that the PT although increased the cell proliferating in the periphery of the lesion site, but due to undesirable microenvironment the neurogenesis decreased concomitantly with functional deficits. This method of PT introduces a reliable, reproducible, noninvasive, and accessible stroke model similar to human stroke condition.